DESCRIPTION OF THE OVERALL PROGRAM (provided by applicant): Our program continues to address the hypothesis that parameters of nuclear structure (chromatin organization and the assembly and activity of nuclear matrix associated subnuclear sites for transcription) contribute to gene expression that mediates the onset, progression and maintenance of bone cell phenotypic properties required for skeletal development and homeostasis. We are pursuing an integrated, team approach that combines molecular, cellular, biochemical and in vivo genetic analyses to define mechanisms by which subnuclear organization of nucleic acids and regulatory proteins facilitates integration of physiological signals to support competency for skeletal gene expression and bone development in vivo. Our program has: 1) demonstrated remodeling of chromatin and nucleosome organization that is linked to developmental induction and steroid hormone enhanced bone specific transcription; 2) identified bone specific activators and co regulators that integrate activities at skeletal gene promoter elements; 3) demonstrated that the Runx2 (Cbfal/AML3) factor is punctately localized at discrete nuclear matrix associated subnuclear sites; and 4) structurally and functionally characterized a unique intranuclear targeting signal that directs Runx2 to sites of transcription as a requirement for skeletal development to proceed in vivo. We will now examine mechanisms that support our fundamental observations that spatial and temporal organization and assembly of regulatory complexes within the nucleus are necessary for expression of genes that control bone formation. Project 1 will investigate mechanisms that direct bone specific transcription factors and coregulatory proteins to sites within the nucleus where developmental and steroid hormone responsive gene expression occurs. Project 2 will determine mechanisms by which the subnuclear organization of Runx2 with coregulatory proteins is functionally coupled to the integration of regulatory cues that control gene expression in the nucleus of osteoblasts and in the multiple nuclei of osteoclasts. Project 3 will pursue mechanisms that control remodeling of chromatin structure of bone specific genes to render promoter elements competent for physiologically responsive regulatory protein interactions. Evaluation of nuclear structure gene expression interrelationships in vitro and in vivo will provide biological validation of regulatory parameters that are obligatory for skeletal development and may be compromised in metabolic bone disease.